The long term objective of this project is to understand how antigens can, depending on circumstances, have two opposing effects on B lymphocytes, on the one hand acting to activate the cell into proliferation and differentiation to end up as an antibody-forming clone; or, on the other hand, to transmit a negative signal to the B cell which may actually kill it or temporarily inactivate it. In other words, we seek to understand the rules of immune induction and tolerance induction. Insights gained will be relevant not only to the design of new vaccination strategies, but also to areas such as autoimmune diseases and organ transplantation. B lymphocytes will be obtained from the spleens and other lymphoid organs of mice. They will be fractionated by flow cytometry and/or antigen affinity procedures into subsets of various sorts, e.g. on the basis of antigenic specificity, idiotypic specificity, immunoglobulin receptor isotype spectrum, intensity of expression of B cell markers and light-scattering characteristics. Selected single B cells will be placed into specialized microcultures and stimulated with antigens, mitogens and interleukins in various combinations. The amount, specificity, and isotype distribution of the antibody formed will be measured by clonal enzyme-linked immunosorbent assays (ELISA). The immunoglobulin V genes expressed by given clones or single cells will be determined by the polymerase chain reaction (PCR) with special emphasis on V gene hypermutations. The helper and suppressor effects of certain T cells on clonal microcultures will be measured. We vill determine the activation requirements of different B cell subsets, such as unimmunized, primed, recirculating, or Ly-1 B cells. We shall study when the somatic V gene hypermutation process so vital to the development of high affinity B cells commences. We will dissect the cellular basis of immunologic tolerance induced in adult mice by soluble, deaggregated antigens exploring the relative contributions of T cell-mediated suppression and direct effects of the antigen in causing anergy in the B cell itself. If antigen-specific suppression is a major contributor to adult tolerance, we shall ask whether the suppressor cell secretes lymphokines which antagonize the activating action of other lymphokines made by helper T cells.